Hyperuricemia is a disease from excessive purine metabolism. Uric acid, the final product of purine metabolism in human bodies, may cause hyperuricemia at a concentration of over 70 mg/L. Depending on its pathogenesis and duration, hyperuricemia may be further classified to acute hyperuricemia and chronic hyperuricemia. Acute hyperuricemia is a common complication in hematologic tumors and treatment therefor. With radio- and chemotherapy against hematologic tumors, urate concentration in vivo may be dramatically elevated by increased cytolysis and purine metabolite release, leading to acute hyperuricemia as well as renal failure in severe cases (Hershfield M S., Cecil Textbook of Medicine (20th), 1508-1515). Hematologic tumor is one of the diseases severely imparing human healths, and common hematologic tumors mainly include various kinds of leukemia, multiple myeloma and malignant lymphoma. As a metabolic disease, chronic hyperuricemia is caused by human purine metabolism disorder in vivo, and is likely to further develop into gout, an acute or chronic disease from the crystallization and precipitation of urates in soft tissues. The major clinical manifestations of gout are repeated attacks of arthritis and/or nephropathia; and meanwhile, with long-term elevation of blood uric acid level, uric acid may be crystallized and precipitated in connective tissues and results in formation of granuloma, which gradually becomes tophus (Nancy J G, Susan J K, Edna S, John S S et al., Arthritis Res Ther., 2005, 8 (1): R12). In the last 20 years, with the improvement of human living standards, the prevalence rate of chronic hyperuricemia/gout rises constantly. An epidemiology survey in England showed that the prevalence rate of gout rises from 1.19% in 1990 to 1.4% in 1999 (Wallace K, Riedel A, Joseph-Ridge N, Wortmann R., J. Rheumatol., 2004, 31: 1582-1587). Another survey in coastal areas of Shandong province, China, in 2008 showed that patients with hyperuricemia take 13.19% part of the total population, whereas patients with gout take 1.14% (Miao Z, Li C, Chen Y, et al., J Rheumatol., 2008; 35: 1859-1864). Therefore, such a disease has become an important one that jeopardizes human health.
Substantially, hyperuricemia is associated with urate oxidase gene mutation and deactivation during human evolutions, wherein a terminator codon is introduced in advance to the coding sequence of urate oxidase (Wu X, Lee C C, Muzny D M, Caskey C T., Proc Natl Acad Sci USA, 1989, 86: 9412-9416), thus disabling the synthesis of active urate oxidase and terminating human purine catabolism at uric acid (Wu X, Muzny D M, Lee C C, Caskey C T., J Mol Evol., 1992, 34: 78-84). Urates with lower solubility (˜11 mg/dl) can be transformed to more soluble allantoin (˜147 mg/dl) by active urate oxidase in liver peroxisomes of non-human primates and other mammals, hence being more effectively excreted by kidney (Wortmann R L, Kelley W N, Kelley's Textbook of Rheumatology (6th), 2001: 1339-1376). On worldwide market, urate oxidase pharmaceuticals include Rasburicase, a recombinant urate oxidase derived from Aspergillus flavus, by Sanofi Inc., France, granted by FDA in 2002 (Bosly A, Sonet A, Pinkerton C R, McCowage G, Bron D, Sanz M A, Van den Berg H., Cancer, 2003, 98: 1048-54). Rasburicase is prepared by Saccharomyces cerevisiae fermentation and purification, and may be used in short-term treatment of severe hyperuricemia from chemotherapy against tumor with better effect than that of allopurinol. However, because the urate oxidase derived from Aspergillus flavus shares a homology of lower than 40% with that from human (Lee C C, Wu X, Gibbs R A, Cook R G Muzny D M, Caskey C T., Science, 1988, 239: 1288-1291), antibodies thereagainst will be generated in vivo in 70% of patients after multiple dosing, thus leading to a rapid decrease of urate oxidase effects as well as severe allergy reactions (Navolanic P M, Pui C-H, Larson R A, et al., Leukemia, 2003, 17: 499-514).
Covalent modification with polyethelene glycol (PEG) has been proved to be useful to decrease protein immunogenicity, increase protein solubility and extend the half-life (Veronese F M, Pasut G, Drug Discov. Today, 2005, 10: 1451-458). Various PEGylated recombinant protein drugs have been granted by FDA at present. To conjugate to a target molecule, PEG needs to be activated at one end or two with a selected functional group, depending on characteristics of molecules to be conjugated. A linking group for PEG covalent modification of protein may be any one of biocompatible linking groups. Common biocompatible linking groups include ester, amido, imido, urethane, succinimidyl (e.g., succinimidyl succinate (SS), succinimidyl propionate (SPA), succinimidyl carboxymethyl (SCM), succinimidyl succinamide (SSA) or N-hydroxyl-succinimidyl (NHS)), epoxyl, oxycarbonyl imidazolyl, nitrophenyl group (e.g., nitrophenyl carbonate (NPC) or trichlorobenzene carbonate (TPC)), and histidinyl or primary amine. Theoretically, the activated PEG may be reacted with main amino acids (e.g., Lys, Cys, His, Asp, Glu, Ser, and Thr, etc.) in a protein at the N-terminus or C-terminus, in which a reaction with Lys at the N-terminus is most commonly used.
PEGylated urate oxidase was studied since the late 1970s. A urate oxidase derived from Candida was modified with cyanuric chloride activated 5 kDa PEG by Nihimura and Tsuji et al. However, with modification rates of up to 20%˜30%, both enzyme activity and retention rate decrease to less than 50%, and immunogenicity is failed to be decreased with sufficient retention on enzyme activity (Nishimura H, Ashihara Y, Matsushima A, Inada Y., Enzyme, 1979, 24: 261-264; Tsuji J-1, Hirose K, Kashara E, Naitoh M, Yamamoto I., Int J. Immunopharmacol., 1985, 7: 725-730). Previously, PEGylated urate oxidase was studied on reaction in human body. A urate oxidase derived from Candida was modified with a 5 kDa PEG by Davis et al. After the conjugate was injected into 5 subjects, the urate oxidase concentration in the serum decreased to be undetectable. 4 weeks later, another injection resulted also in undetectable urate oxidase antibodies, as detected by gel diffusion method that is not so sensitive (Davis F F, Abuchowshi A, Karp D., J Pharmacol Exp Ther., 1981, 219: 352-354). From the foregoing, the PEGylated urate oxidase is safe and effective in human body. However, there are problems on long-term security and effectiveness, owing to high antigenicity of urate oxidase and deficiency in PEG agents.
With the appearance of the second generation of branch-chained PEG with high molecular weight, PEGylated urate oxidase was studied again. A recombinant swine-gelada urate oxidase was modified with 10 kDa mPEG-NPC at Lys residue by Duke University and Savient Inc. (Pegloticase) (Michael H, Susan J. K., 2006, U.S. Pat. No. 7,056,713B1). With each subunit conjugated with 9 PEGs, the enzyme activity may be retained by more than 75%, and the immunogenicity is substantially eliminated as verified by animal experiments. (Sherman M R, Saifer M G, Perez-Ruiz F., Adv Drug Deliv Rev., 2008, 60: 59-68). Pegloticase has been granted by FDA at Sep. 14, 2010 for long-term treatment (6 months) of refractory gout. However, antibodies against Pegloticase occur in 90% of clinical tests, and the drug is only suitable for intravenous injection. Because of the potential immunogenicity and inconveniency for administration, long-term treatment obedience in patients with chronic gout is decreased.
Drug development on PEGylated urate oxidase was also carried out by Phoenix Inc. (Ensor C M, Clark M A, Holtsberg F W., 2005, U.S. Pat. No. 6,913,915B2), the urate oxidase modified by whom is a Candida urate oxidase, with preferred PEG as mPEG-SC and mPEG-SPA with an average molecular weight of 20 KD, and modified site as the same Lys residue. Each protein was conjugated with 18˜22 PEG chains. The modified urate oxidase retains 75% of activity with a half-life of up to 3 days in mice body, far longer than that of non-modified urate oxidase, namely, 4 hours (Bomalaski J S, Holtsberg F W, Ensor C M, Clark M A., J Rheumatol., 2002, 29: 1942-1949). Said PEGylated urate oxidase was studied clinically at 2001. However, phase I clinical trial showed that the efficacy is weakened after administration, and thus the research was terminated (Bolmalaski J S, Goddard D H, Grezlak D, et al., Arthritis Rheum., 2002, 46: S141).
So far, there is worldwidely no commercialized urate oxidase product that has an appropriate half-life and may be used in long-term treatment with security and no immunogenicity. Although the human urate oxidase has become a pseudogene because of the mutation and loss of activity, the immunogenicity of the enzyme would be reduced if the gene was reformed and the activity was recovered. However, because of missense mutations accumulated during the evolution due to the lack of selective pressure, it is difficult to find out and correct all mutations and recover the human uricase activity. Because of the high activity of microorganism urate oxidases and the low immunogenicity of mammal urate oxidases, the two urate oxidases are studied for long-term use of urate oxidase at now. Though PEGylation may decrease the immunogenicity of microorganism derived urate oxidase, the immunoreaction is not effectively eliminated in human bodies. Clinical research on long-term treatment of chronic gout with the PEGylated recombinant microorganism urate oxidase has been stopped in foreign countries (such as in Phenix Inc. and Enzon Inc.). In contrast, in mammal urate oxidase studies, PEGylated swine urate oxidase from Duke University and Savient Inc. has successfully come into market, indicating that protein homology is still a key element for the immunogenicity of PEGylated proteins. To further improve the protein homology of urate oxidase, the inventor has carried out a study on human-canine chimeric urate oxidase on the basis of canine urate oxidase, which exhibits higher homology to humanized urate oxidase and greater specific activity, for which a Chinese patent application (No. 200910191240.3) and a PCT application (No. PCT/CN2010/071020) have been filed, respectively. PEGylation on the basis of the above canine urate oxidase analogue is studied in the present invention for a new and long-acting recombinant urate oxidase, which, as retaining high enzymatic activity, may be possessed with extended half-life and decreased immunogenicity in vivo for the treatment of acute hyperuricemia from chemotherapy against tumors and hyperuricemia or chronic gout from metabolic disorders.